Galactoside inhibitor of galectins

ABSTRACT

Provided is a compound of the general formula (I): 
     
       
         
         
             
             
         
       
     
     The compound of formula (I) is suitable for treating pulmonary fibrosis, such as Idiopathic pulmonary fibrosis in a mammal. Also provided is a method for treatment of pulmonary fibrosis, such as Idiopathic pulmonary fibrosis in a human subject having a galectin-3 level indicative of pulmonary fibrosis or exacerbation of symptoms as well as a method for making said compound.

TECHNICAL FIELD

The present invention relates to novel compounds, the use of saidcompounds as medicament and for the manufacture of a medicament for thetreatment of pulmonary fibrosis, such as Idiopathic pulmonary fibrosisin mammals. The invention also relates to pharmaceutical compositionscomprising said novel compounds. Furthermore the present inventionrelates to pulmonary administration, in particular the use of nebulizersfor providing optimal treatment of IPF. The present invention alsorelates to a method of monitoring development or progression ofpulmonary fibrosis as well as a method of monitoring or predictingexacerbation of symptoms.

BACKGROUND ART

Idiopathic pulmonary fibrosis (IPF) represents a massive worldwidehealth burden. It is a chronic condition of unknown etiology in whichrepeated acute lung injury causes progressive fibrosis resulting indestruction of lung architecture, deteriorating lung function withconsequent respiratory failure and death. Although idiopathic pulmonaryfibrosis (IPF) is the archetypal and most common cause of lung fibrosis,numerous respiratory diseases can progress to pulmonary fibrosis, andthis usually signifies a worse prognosis. The median time to death fromdiagnosis is 2.5 years and the incidence and prevalence of IPF continuesto rise. It remains one of the few respiratory conditions for whichthere are no effective therapies, and there are no reliable biomarkersto predict disease progression. The mechanisms resulting in pulmonaryfibrosis are unclear but centre around aberrant wound healing as aconsequence of repetitive epithelial injury from an as yet unknowncause. IPF is characterized by fibroblastic foci containingfibroblasts/myofibroblasts which show increased activation response tofibrogenic cytokines such as transforming growth factor-β1 (TGF-β1).Given the non-responsiveness of many cases of IPF to currentanti-inflammatory treatments the myofibroblasts within fibroblastic focirepresent a potential novel therapeutic target. There is a big unmetneed for drugs for treatment of Idiopathic pulmonary fibrosis.

The bleomycin model of pulmonary fibrosis is the best characterizedrodent model and is the industry standard model. Bleomycin treatmentcauses oxidant-mediated DNA damage and induces initial lung inflammationfollowed by progressive fibrosis over 2-4 weeks. When administeredduring the later phase of the injury the anti-fibrotic potential ofnovel compounds can be assessed.

Galectin inhibitors, in particular Gal-3 inhibitors have been describedby the some of the present inventors in earlier published patentapplications. None of these galectin inhibitors have been tested in ableomycin model. Some of the prior art galectin inhibitors have thefollowing general formulas

as described in WO/2005/113568,and

as described in WO/2005/113569, in which Fe can be a D-galactose,and

as described in WO/2010/126435.

Furthermore there is a big need for bio-markers for making it lesscomplicated to perform clinical trials in patients with pulmonaryfibrosis. No biomarkers exist that are suitable for detection ofpatients with pulmonary fibrosis or specifically idiopathic pulmonaryfibrosis. Similarly, no biomarkers are suitable for prediction of theprognosis for patients with pulmonary fibrosis, for identification ofpatients with mild or aggressive forms of the disease, foridentification of patients with ongoing or preeminent exacerbations, andfor tracking the development of the patient's disease level. This makesit very complicated and costly to perform clinical trials of noveltreatments in these patients.

SUMMARY OF THE DISCLOSURE

Galectin-3 is a β-galactoside binding lectin that is highly expressed infibrotic tissue of diverse etiologies. The role of galectin-3 inbleomycin and TGF-β1-induced lung fibrosis in mice is examined, and itsrelevance in human IPF is established. Studies with galectin-3 aredescribed in MacKinnon et al., “Regulation of TGF-β1 driven lungfibrosis by galectin-3”, Am. J. Respir. Crit. Care Med. 185: 537-546(2012, originally available online on Nov. 17, 2011).). In particular,it is shown that galectin-3 inhibition may represent a novel therapeuticstrategy for treatment of lung fibrosis. A novel compound has beentested and shown to be an inhibitor of galectin-3, in particular, thiscompound blocked TGF-β-induced β-catenin activation in vitro andattenuated the late stage progression of lung fibrosis followingbleomycin in vivo.

The publications and other materials, including patents, used herein toillustrate the invention and, in particular, to provide additionaldetails respecting the practice are incorporated by reference in theirentirety.

Accordingly, provided is a compound of the general formula (I):

In a further aspect, provided is a composition, particularly, apharmaceutical composition comprising the compound of formula (I) andoptionally a pharmaceutically acceptable additive, such as carrier orexcipient.

The compound of formula (I) is suitable for use in a method for treatingpulmonary fibrosis, such as Idiopathic pulmonary fibrosis in a mammal.Typically, such mammal is a human subject. The mode of administration istypically selected from oral, intra venous (i.v.), subcutaneous (s.c.),and pulmonary. In particular the pulmonary route has been shown toprovide a considerably longer half-life than the i.v. or s.c. routes inmice. When treating pulmonary fibrosis, in particular IPF, it isimportant to obtain adequately high local concentrations of thetherapeutic in the narrowest parts of the lung tissue, including thebronchioles and the alveoli. Further, it is important that thetherapeutic obtains an adequate residence time at the site of action inthe lung tissue. Furthermore, since the fibrosis in IPF patients is onlylocated in the lung, it is preferable to obtain a high lung exposurewith minimal or no systemic exposure and the use of nebulizers inparticular electronic nebulizers of the ultrasonic type is effective.However, cough is a central symptom for patients with pulmonary fibrosisand in particular IPF—a symptom that is likely to be aggravated if anirritant is introduced into the lung. Hence, treatment with a drypowder, such as with a dry powder inhaler or similar, is not suitablefor these patients. However, delivering the compound using a nebulizer,such as an electronic nebulizer, is particularly beneficial, since itallows delivery of the compound to the smallest compartments in thelung, without causing any irritation in the lung.

Moreover, in a still further aspect provided is a method for treatmentof pulmonary fibrosis, such as Idiopathic pulmonary fibrosis comprisingadministering to a mammal in need thereof a therapeutically effectiveamount of the compound of formula (I).

In another aspect, provided is a process of preparing a compound offormula I comprising the step of reactingbis-(3-deoxy-3-azido-β-D-galactopyranosyl) sulfane with3-fluorophenylacetylene and an amine, such as triethylamine, optionallyin the presence of a catalyst, such as Cu(I), in a solvent, such asN,N-dimethylformamide (DMF), resulting in the compound of formula I.

Moreover, the present inventors have discovered that concentrations ofthe human protein galectin-3 in body fluids or tissues of a humansubject can be used to for instance predict or monitor diseaseprogression or therapeutic efficacy in human subjects with pulmonaryfibrosis.

Measurement of galectin-3 levels in lung tissue, broncho-alveolar lavage(BAL) fluid, blood, serum or plasma can be used to identify patientswith lung fibrosis and to assess the severity of the disease.Longitudinal studies of the levels of galectin-3 in lung tissue,broncho-alveolar lavage fluid, blood, serum or plasma can be used topredict exacerbations and to follow the development of the disease.Hence, measurement of galectin-3 levels in lung tissue, broncho-alveolarlavage fluid, blood, serum or plasma before, during and after completionof a treatment for pulmonary fibrosis can be used to assess the effectof such treatment.

The results of the present inventors demonstrate that serum galectin-3levels may help distinguish patients with usual interstitial pneumonia(UIP) from patients with non-specific interstitial pneumonia (NSIP) andidentify patients undergoing an acute exacerbation of their IPF.

In a further aspect the present invention relates to a method ofdiagnosing pulmonary fibrosis in a human subject comprising a) measuringa galectin-3 level (e.g. concentration) in a body sample from the humansubject using a suitable test method, b) comparing the galectin-3 levelto a predetermined reference level, and c) determining whether thegalectin-3 level is indicative of diagnosing the subject with pulmonaryfibrosis.

In a still further aspect the present invention relates to a method ofpredicting the prognosis pulmonary fibrosis in a human subjectcomprising a) measuring a galectin-3 level (e.g. concentration) in abody sample from the human subject using a suitable test method, and b)determining whether the galectin-3 level is indicative of a poorprognosis or not for the human subject.

In a further aspect the present invention relates to a method ofmonitoring development or progression of pulmonary fibrosis in a humansubject, comprising a) measuring a galectin-3 level in a body samplefrom the subject at least two times with sufficient interval(s) tomeasure a clinically relevant change, b) comparing the galectin-3 levelto a predetermined reference level, and repeating steps a) and b) one ormore times to monitor the development or progression of pulmonaryfibrosis in the human subject.

In a still further aspect the present invention relates to a method ofmonitoring or predicting exacerbation of symptoms in a human subjectwith pulmonary fibrosis comprising a) measuring a galectin-3 level (e.g.concentration) in a body sample from the human subject using a suitabletest method, b) comparing the galectin-3 level to a predeterminedreference level, and c) determine the presence or absence of agalectin-3 level indicative of the development or progression ofexacerbation of symptoms.

In a further aspect the present invention relates to a method fortreatment of pulmonary fibrosis, such as Idiopathic pulmonary fibrosis,in a human subject having a galectin-3 level indicative of pulmonaryfibrosis or exacerbation of symptoms comprising administering to a humansubject a therapeutically effective amount of a galectin-3 inhibitor.

Any one of the above methods can include the step of transmitting,displaying, storing, or printing; or outputting to a user interfacedevice, a computer readable storage medium, a local computer system or aremote computer system, information related to the likelihood ofdeveloping pulmonary fibrosis in the subject or for characterization ofthe degree of severity of the pulmonary fibrosis in said subject.

Accordingly, disclosed herein, inter alia are the following embodiments:

1. A compound of the general formula (I):

2. The compound of embodiment 1 selected frombis(3-deoxy-3-(3-fluorophenyl-1H-1,2,3-triazol-1-yl)-β-D-galactopyranosyl)sulfane as the free form.

3. The compound according to any one of embodiments 1-2, for use as amedicament.

4. A pharmaceutical composition comprising the compound of any one ofembodiments 1-3 and optionally a pharmaceutically acceptable additive,such as a carrier or an excipient.

5. The pharmaceutical composition of embodiment 4 wherein thecomposition is administered by the pulmonary route.

6. The pharmaceutical composition of embodiment 5 wherein administrationby the pulmonary route is selected from a nebulizer such as anultrasonic nebulizer or a jet nebulizer.

7. The compound of any one of the embodiments 1-3 for use in a methodfor treating pulmonary fibrosis, such as Idiopathic pulmonary fibrosisin a mammal.

8. The compound of embodiment 7, wherein the compound is administered bythe pulmonary route.

9. The compound of embodiment 8, wherein administration by the pulmonaryroute is selected from a nebulizer such as an ultrasonic nebulizer or ajet nebulizer.

10. The compound of embodiment 7, 8 or 9 wherein said mammal is a humansubject.

11. A method for treatment of pulmonary fibrosis, such as Idiopathicpulmonary fibrosis comprising administering to a mammal in need thereofa therapeutically effective amount of the compound of any one ofembodiments 1-3.

12. The method of embodiment 11, wherein the compound of any one ofembodiments 1-3 is administered by the pulmonary route.

13. The method of embodiment 12, wherein administration by the pulmonaryroute is selected from a nebulizer such as an ultrasonic nebulizer or ajet nebulizer.

14. A process of preparing a compound of formula I comprising a step ofreacting bis-(3-deoxy-3-azido-β-D-galactopyranosyl) sulfane with3-fluorophenylacetylene and an amine in a solvent, resulting in thecompound of formula I.

15. The process of embodiment 14 wherein the amine is triethylamine, acatalyst is present, such as Cu(I), and the solvent is an organicsolvent, such as N,N-dimethylformamide (DMF).

16. A nebulizer device for pulmonary administration comprising acompound of any one of embodiments 1-3.

17. The nebulizer device of embodiment 16, wherein the compound isbis(3-deoxy-3-(3-fluorophenyl-1H-1,2,3-triazol-1-yl)-β-D-galactopyranosyl)sulfane as the free form.

18. The nebulizer of embodiment 16 or 17 which is selected from anultrasonic nebulizer or a jet nebulizer.

19. A dry powder device for pulmonary administration comprising acompound of any one of embodiments 1-3.

20. The dry powder device of embodiment 19, wherein the compound isbis(3-deoxy-3-(3-fluorophenyl-1H-1,2,3-triazol-1-yl)-β-D-galactopyranosyl)sulfane as the free form.

21. A method of diagnosing pulmonary fibrosis in a human subjectcomprising a) measuring a galectin-3 level (e.g. concentration) in abody sample from the human subject using a suitable test method, b)comparing the galectin-3 level to a predetermined reference level, andc) determining whether the galectin-3 level is indicative of diagnosingthe subject with pulmonary fibrosis.

22. The method of embodiment 21 wherein the indicative level ofgalectin-3 is at least 22 ng/ml, such as at least 25 ng/ml, such as atleast 30 ng/ml, at least 40 ng/ml, at least 50 ng/ml, at least 60 ng/ml,at least 70 ng/ml.

23. A method of predicting the prognosis of pulmonary fibrosis in ahuman subject comprising a) measuring a galectin-3 level (e.g.concentration) in a body sample from the human subject using a suitabletest method, and b) determining whether the galectin-3 level isindicative of a poor prognosis or not for the human subject.

24. The method of embodiment 23 wherein the indicative level ofgalectin-3 is at least 22 ng/ml, such as at least 25 ng/ml, such as atleast 30 ng/ml, at least 40 ng/ml, at least 50 ng/ml, at least 60 ng/ml,at least 70 ng/ml.

25. A method of monitoring development or progression of pulmonaryfibrosis in a human subject, comprising a) measuring a galectin-3 levelin a body sample from the subject at least two times with sufficientinterval(s) to measure a clinically relevant change, b) comparing thegalectin-3 level to a predetermined reference level, and repeating stepsa) and b) one or more times to monitor the development or progression ofpulmonary fibrosis in the human subject.

26. The method of embodiment 25 wherein the time period between twomeasurements is independently selected from about 2 weeks to 2 years,such as 2 weeks, 4 weeks, 1 month, 2 months, 3 months 6 months, 1 year,or 2 years.

27. The method of embodiment 25 wherein when the indicative level ofgalectin-3 is below 22 ng/ml treatment of pulmonary fibrosis may bestopped, adjusted or put on hold.

28. The method of embodiment 25 wherein when the indicative level ofgalectin-3 is at least 22 ng/ml, such as at least 25 ng/ml, such as atleast 30 ng/ml, at least 40 ng/ml, at least 50 ng/ml, at least 60 ng/ml,at least 70 ng/ml treatment of pulmonary fibrosis may be initiated orincreased.

29. A method of monitoring or predicting exacerbation of symptoms in ahuman subject with pulmonary fibrosis comprising a) measuring agalectin-3 level (e.g. concentration) in a body sample from the humansubject using a suitable test method, b) comparing the galectin-3 levelto a predetermined reference level, b) determine the presence or absenceof a galectin-3 level indicative of the development or progression ofexacerbation of symptoms, and c) repeating steps a) and b) to monitor orpredict the development or progression of the exacerbation of symptomsin the human subject.

30. The method of embodiment 29 wherein when the indicative level ofgalectin-3 is below 22 ng/ml treatment of pulmonary fibrosis may bestopped, adjusted or put on hold.

31. The method of embodiment 29 wherein when the indicative level ofgalectin-3 is at least 22 ng/ml, such as at least 25 ng/ml, such as atleast 30 ng/ml, at least 40 ng/ml, at least 50 ng/ml, at least 60 ng/ml,at least 70 ng/ml treatment of pulmonary fibrosis is initiated orincreased. 32. The method of embodiment 29 wherein when the indicativelevel of galectin-3 is at least 50 ng/ml, at least 60 ng/ml, at least 70ng/ml prophylactic treatment of exacerbation of symptoms is initiated orincreased.

33. The method of any one of embodiments 17-28 wherein the pulmonaryfibrosis is idiopathic pulmonary fibrosis.

34. The method of any one of embodiments 21-33 wherein the subject isdiagnosed with mild, moderate or aggressive forms of pulmonary fibrosisaccording to the level of galectin-3.

35. The method of any one of embodiments 21-34 wherein in step a)further bio-markers are measured which markers are relevant forpulmonary fibrosis, including markers linked to Galectin-3 levels,leading to a more accurate diagnosis, prognosis, and/or monitoring.

36. The method of embodiment 35 wherein the bio-markers are selectedfrom MMP7, perDLCO, KL-6, SP-A, MMP-7, CCL-18, IL13, CC-chemokines,IL10, IL1 receptor antagonist, CCL2, Calgranulin B (S100A9 or MRP14),macrophage migration inhibitory factor (MIF), pro-collagen, pro-collagen3.

37. The method of embodiment 35 wherein the bio-markers are selectedfrom analysis of the presence and frequency of certain cell types inbody fluids from said human subject.

38. The method of embodiment 36 wherein the bio-markers are selectedfrom analysis of the presence and frequency of fibrocytes and T-cellsubpopulations in body fluids from said human subject.

39. The method of any one of embodiments 21-38 wherein the predeterminedreference level for galectin-3 is in the range from about 10.0 ng/mL toabout 25.0 ng/mL, such as in the range from about 13.0 ng/mL to about19.2 ng/mL.

40. The method of any one of embodiments 21-39 wherein the body sampleis selected from blood, serum, plasma, broncho-alveolar lavage fluid,lung tissue.

41. The method of any one of embodiments 21-40 wherein the suitable testmethod is selected from an immunoassay, an immunohistochemical assay, acolorimetric assay, a turbidimetric assay, and flow cytometry.

42. The method of embodiment 17 or 29, wherein the subject has agalectin-3 blood concentration determined to be within a target range.

43. The method of embodiment 42, wherein the target range is from about10 ng/ml to about 70 ng/ml.

44. A method for treatment of pulmonary fibrosis, such as Idiopathicpulmonary fibrosis in a human subject having a galectin-3 levelindicative of pulmonary fibrosis or exacerbation of symptoms comprisingadministering to a human subject a therapeutically effective amount of agalectin-3 inhibitor.

45. The method of embodiment 40 wherein the galectin-3 inhibitor isselected from the compound of any one of embodiments 1-3.

46. The method of embodiment 44 wherein the indicative level ofgalectin-3 is at least 22 ng/ml, such as at least about 25 ng/ml, suchas at least about 30 ng/ml, at least about 40 ng/ml, at least about 50ng/ml, at least about 60 ng/ml, at least about 70 ng/ml.

47. The method of embodiment 44 comprising the additional step ofmonitoring the subject's galectin-3 blood level after the therapy isinitiated.

Accordingly, also disclosed herein, inter alia are the followingembodiments:

1. A compound of the general formula (I):

2. The compound of embodiment 1 selected frombis(3-deoxy-3-(3-fluorophenyl-1H-1,2,3-triazol-1-yl)-β-D-galactopyranosyl)sulfane as the free form.

3. A composition comprising the compound of embodiment 1 and optionallya pharmaceutically acceptable additive.

4. A method for treatment of pulmonary fibrosis comprising administeringto a mammal in need thereof an amount of the compound of embodiment 1effective to treat said pulmonary fibrosis.

5. The method of embodiment 4, wherein said compound is administered bythe pulmonary route.

6. A process of preparing a compound of formula I comprising a step ofreacting bis-(3-deoxy-3-azido-β-D-galactopyranosyl) sulfane with3-fluorophenylacetylene and an amine in a solvent, resulting in thecompound of formula I.

7. The process of embodiment 5, wherein the amine is triethylamine, acatalyst is present, such as Cu(I), and the solvent is an organicsolvent, such as N,N-dimethylformamide (DMF).

8. A device for pulmonary administration, wherein said device is anebulizer or dry powder device comprising the compound of embodiment 1.

9. The device of embodiment 8, wherein said device is a nebulizer whichis selected from an ultrasonic nebulizer or a jet nebulizer.

10. A method of detecting the presence and/or extent of pulmonaryfibrosis to diagnose pulmonary fibrosis and/or predict the prognosis ofpulmonary fibrosis in a human subject comprising a) measuring agalectin-3 level in a body sample from the human subject using asuitable test method, b) comparing the galectin-3 level to apredetermined reference level, and c) determining whether the galectin-3level is indicative of diagnosing the subject with pulmonary fibrosis.

11. The method of embodiment 10, wherein the indicative level ofgalectin-3 is at least about 22 ng/ml.

12. A method of monitoring development or progression of or exacerbationof symptoms of pulmonary fibrosis in a human subject, comprising a)measuring a galectin-3 level in a body sample from the subject at leasttwo times with sufficient interval(s) to measure a clinically relevantchange, b) comparing the galectin-3 level to a predetermined referencelevel, and repeating steps a) and b) one or more times to monitor thedevelopment or progression of pulmonary fibrosis in the human subject.

13. The method of embodiment 12, wherein the time period between twomeasurements is independently selected from about 2 weeks to about 2years.

14. The method of embodiment 12, wherein when the level of galectin-3 isbelow about 22 ng/ml treatment of pulmonary fibrosis is stopped,adjusted or put on hold.

15. The method of embodiment 12, wherein the pulmonary fibrosis isidiopathic pulmonary fibrosis.

16. The method of embodiment 12, wherein the subject is diagnosed withmild, moderate or aggressive forms of pulmonary fibrosis according tothe level of galectin-3.

17. The method embodiment 12, wherein in step a) further bio-markers aremeasured, where said markers are linked to Galectin-3 levels.

18. The method of embodiment 17, wherein the bio-markers are selectedfrom MMP7, perDLCO, KL-6, SP-A, MMP-7, CCL-18, IL13, CC-chemokines,IL10, IL1 receptor antagonist, CCL2, Calgranulin B (S100A9 or MRP14),macrophage migration inhibitory factor (MIF), pro-collagen, pro-collagen3.

19. The method of embodiment 17, wherein the bio-markers are selectedfrom analysis of the presence and frequency of certain cell types inbody fluids from said human subject.

20. The method of embodiment 17, wherein the bio-markers are selectedfrom analysis of the presence and frequency of fibrocytes and T-cellsubpopulations in body fluids from said human subject.

21. The method of embodiment 12, wherein the predetermined referencelevel for galectin-3 is in the range from about 10.0 ng/mL to about 25.0ng/mL.

22. The method of embodiment 12, wherein the body sample is selectedfrom blood, serum, plasma, broncho-alveolar lavage fluid, lung tissue.

23. The method of embodiment 10, wherein the suitable test method isselected from an immunoassay, an immunohistochemical assay, acolorimetric assay, a turbidimetric assay, and flow cytometry.

24. The method of embodiment 10, wherein the subject has a galectin-3blood concentration determined to be within a target range from about 10ng/ml to about 70 ng/ml.

25. The method of embodiment 12, wherein the subject has a galectin-3blood concentration determined to be target range is from about 10 ng/mlto about 70 ng/ml.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows (A) Total lung collagen measured by Sircol assay; (B)Fibrosis score; and (C) Inflammatory score. Results represent the meanand SEM (A) or box and whiskers (median, interquartile range, minimum tomaximum, B and C) of n=8 mice per group (n=7 bleo). ***P<0.005,**P<0.01, *P<0.05. FIG. 1D shows the effect of treatment of primaryalveolar epithelial cells from WT mice with TGF-β1 in the presence orabsence of 10 μM TD139 on active β-catenin, total β-catenin and β-actinusing western blot analysis. In FIG. 1E, beta-catenin activation in vivowas assessed by scoring sections of bleomycin treated mouse lung(control and 10 ug TD139 treated) stained with an anti-active betacatenin.

DETAILED DESCRIPTION

In a broad aspect, provided is a compound of the general formula (I):

The compound of formula (I) has the chemical name (IUPAC)bis(3-deoxy-3-(3-fluorophenyl-1H-1,2,3-triazol-1-yl)-β-D-galactopyranosyl)sulfane, and as used herein is intended to cover the compound of formula(I) in any possible form, such as solid or liquid, a salt, a solvate, orin free form.

Typically, the compound of formula (I) isbis(3-deoxy-3-(3-fluorophenyl-1H-1,2,3-triazol-1-yl)-β-D-galactopyranosyl)sulfane as the free form. In a further embodiment the compound offormula (I) isbis(3-deoxy-3-(3-fluorophenyl-1H-1,2,3-triazol-1-yl)-β-D-galactopyranosyl)sulfane as the free form without any solvate, such as anhydrated.

In a further embodiment the compound of formula (I) isbis(3-deoxy-3-(3-fluorophenyl-1H-1,2,3-triazol-1-yl)-β-D-galactopyranosyl)sulfane on amorphous form.

In a further embodiment the compound of formula (I) isbis(3-deoxy-3-(3-fluorophenyl-1H-1,2,3-triazol-1-yl)-β-D-galactopyranosyl)sulfane on a crystalline form.

In a still further embodiment, the compound of formula (I) is useful fortreating pulmonary fibrosis, and therefore is suitable for use as amedicament.

In a further aspect, provided is a compound of formula (I) for use in amethod for treating pulmonary fibrosis, such as Idiopathic pulmonaryfibrosis in a mammal. Such a mammal is typically a human subject,preferably a human subject diagnosed with IPF.

In a still further aspect, provided is a method for treatment ofpulmonary fibrosis, such as Idiopathic pulmonary fibrosis comprisingadministering to a mammal a therapeutically effective amount of acompound of formula (I).

When the compounds and pharmaceutical compositions herein disclosed areused for the above treatment, a therapeutically effective amount of atleast one compound is administered to a mammal in need of saidtreatment.

The term “treatment” and “treating” as used herein means the managementand care of a patient for the purpose of combating a condition, such asa disease or a disorder. The term is intended to include the fullspectrum of treatments for a given condition from which the patient issuffering, such as administration of the active compound to alleviatethe symptoms or complications, to delay the progression of the disease,disorder or condition, to alleviate or relief the symptoms andcomplications, and/or to cure or eliminate the disease, disorder orcondition as well as to prevent the condition, wherein prevention is tobe understood as the management and care of a patient for the purpose ofcombating the disease, condition, or disorder and includes theadministration of the active compounds to prevent the onset of thesymptoms or complications. The treatment may either be performed in anacute or in a chronic way. The patient to be treated is preferably amammal; in particular a human being, but it may also include animals,such as dogs, cats, cows, sheep and pigs.

The term “a therapeutically effective amount” of a compound of formula(I) of the present invention as used herein means an amount sufficientto cure, alleviate or partially arrest the clinical manifestations of agiven disease and its complications. An amount adequate to accomplishthis is defined as “therapeutically effective amount”. Effective amountsfor each purpose will depend on the severity of the disease or injury aswell as the weight and general state of the subject. It will beunderstood that determining an appropriate dosage may be achieved usingroutine experimentation, by constructing a matrix of values and testingdifferent points in the matrix, which is all within the ordinary skillsof a trained physician or veterinary.

In a still further aspect the present invention relates to apharmaceutical composition comprising the compound of formula (I) andoptionally a pharmaceutically acceptable additive, such as a carrier oran excipient.

As used herein “pharmaceutically acceptable additive” is intendedwithout limitation to include carriers, excipients, diluents, adjuvant,colorings, aroma, preservatives etc. that the skilled person wouldconsider using when formulating a compound of the present invention inorder to make a pharmaceutical composition.

The adjuvants, diluents, excipients and/or carriers that may be used inthe composition of the invention must be pharmaceutically acceptable inthe sense of being compatible with the compound of formula (I) and theother ingredients of the pharmaceutical composition, and not deleteriousto the recipient thereof. It is preferred that the compositions shallnot contain any material that may cause an adverse reaction, such as anallergic reaction. The adjuvants, diluents, excipients and carriers thatmay be used in the pharmaceutical composition of the invention are wellknown to a person within the art.

As mentioned above, the compositions and particularly pharmaceuticalcompositions as herein disclosed may, in addition to the compoundsherein disclosed, further comprise at least one pharmaceuticallyacceptable adjuvant, diluent, excipient and/or carrier. In someembodiments, the pharmaceutical compositions comprise from 1 to 99weight % of said at least one pharmaceutically acceptable adjuvant,diluent, excipient and/or carrier and from 1 to 99 weight % of acompound as herein disclosed. The combined amount of the activeingredient and of the pharmaceutically acceptable adjuvant, diluent,excipient and/or carrier may not constitute more than 100% by weight ofthe composition, particularly the pharmaceutical composition.

In some embodiments, only one compound as herein disclosed is used forthe purposes discussed above.

In some embodiments, two or more of the compound as herein disclosed areused in combination for the purposes discussed above.

The composition, particularly pharmaceutical composition comprising acompound set forth herein may be adapted for oral, intravenous, topical,intraperitoneal, nasal, buccal, sublingual, or subcutaneousadministration, or for administration via the respiratory tract in theform of, for example, an aerosol or an air-suspended fine powder.Therefore, the pharmaceutical composition may be in the form of, forexample, tablets, capsules, powders, nanoparticles, crystals, amorphoussubstances, solutions, transdermal patches or suppositories.

Thus, in a still further aspect provided is a composition, particularlya pharmaceutical composition for intrapulmonary administration.Typically, such composition is delivered by a nebulizer or inhaler,preferably a nebulizer.

Boehringer Ingelheim provided a new technology in 1997 named Raspimatwhich is a mechanical nebulizer of the soft mist inhaler type. Thismechanical nebulizer is operated by hand without any need for a gaspropellant and no need for electrical power. Another mechanicalnebulizer is a human powered nebulizer developed by a team fromMarquette University. This nebulizer can by operated by an electricalcompressor, but it is also suitable for simple mechanical pumps in orderto provide a mist into the lungs of patients. Further nebulizers of theelectrical type are ultrasonic nebulizers based on the vibrating meshtechnology developed by inter alia PARI, Respironics, Omron, Beurer,Aerogen, or ultrasonic nebulizers based on an electronic oscillator thatgenerate a high frequency ultrasonic wave developed by inter alia Omronand Beurer. A further electrical nebulizer is a jet nebulizer also knownas atomizers.

In a further embodiment the nebulizer is selected from a mechanicalnebulizer, such as a soft mist inhaler or a human powered nebulizer. Inanother embodiment the nebulizer is selected from an electricalnebulizer, such as a nebulizer based on ultrasonic vibrating meshtechnology, a jet nebulizer, or an ultrasonic wave nebulizer. Particularsuitable nebulizers are based on vibrating mesh technology such as eFlowfrom PARI. When treating pulmonary fibrosis, in particular IPF, it isimportant to obtain adequately high local concentrations of thetherapeutic in the narrowest parts of the lung tissue, including thebronchioles and the alveoli. Further, it is important that thetherapeutic obtains an adequate residence time at the site of action inthe lung tissue. However, cough is a central symptom for patients withpulmonary fibrosis and in particular IPF—a symptom that is likely to beaggravated if an irritant is introduced into the lung. Hence, treatmentwith a dry powder, such as with a dry powder inhaler or similar, is notsuitable for these patients. However, delivering the compound using anebulizer, such as an electronic nebulizer, is particularly beneficial,since it allows delivery of the compound to the smallest compartments inthe lung, without causing any irritation in the lung. Such relevantnebulizer systems are described in published patent applicationsUS20040089295, US20050056274, US20060054166, US20060097068,US20060102172, US20080060640, US20110155768, and US20120167877, all ofwhich are incorporated herein by reference. Other suitable nebulizersare Tyvaso inhalation system from United Therapeutics, Allera nebulizersystem from Gilead, Bronchitol inhaler from Pharmaxis, Diskhaler fromGSK, jet and ultrasonic nebulizers from Actelion and Profile Pharma.

The following characteristics are required for the pulmonary deliverydevice: It should be able to provide a specific dose accurately andrepeatedly. It should be able to provide 2 or more different doselevels, for instance through repeated dosing or by adjusting the doseprovide to the patient. The device should ensure that the drug isdelivered to the bronchiolar space or preferably to the bronchiolar andthe alveolar space of the lung preferably uniformly over the lungtissue. Hence, the device should generate aerosols or dry powder of anadequately small size to ensure this delivery, while not deliveringparticles so small that they are immediately exhaled and thus notremaining in the lung tissue.

Inhalation nebulizers deliver therapeutically effective amounts ofpharmaceuticals by forming an aerosol which includes particles of a sizethat can easily be inhaled. The aerosol can be used, for example, by apatient within the bounds of an inhalation therapy, whereby thetherapeutically effective pharmaceutical or drug reaches the patient'srespiratory tract upon inhalation.

A variety of inhalation nebulizers are known. EP 0 170 715 A1 uses acompressed gas flow to form an aerosol. A nozzle is arranged as anaerosol generator in an atomizer chamber of the inhalation nebulizer andhas two suction ducts arranged adjacent a compressed-gas channel. Whencompressed air flows through the compressed-gas channel, the liquid tobe nebulized is drawn in through the suction ducts from a liquid storagecontainer.

EP 0 432 992 A discloses a nebulizer comprising an aerosol generatorhaving a liquid storage container, a perforate membrane and a vibrator.The vibrator is operable to vibrate the membrane such that it dispensesan aerosol from a liquid through holes provided in the membrane.

U.S. Pat. No. 5,918,593 relates to ultrasonic nebulizers generating anaerosol by interaction between an amount of liquid and a piezo electricelement. Droplets of various sizes are expelled from a surface of aliquid bulk when vibrational energy is transferred from the piezoelement to the liquid. The droplets thus generated are filtered in anatomizer chamber since oversized droplets have to be removed from thedroplets expelled from the surface in order to generate an aerosol forinhalation by a patient. This nebulizer is representative ofcontinuously operating inhalation nebulizers, in which the aerosolgenerator produces an aerosol not only during inhalation but also whilethe patient exhales. The aerosol produced by the aerosol generator isactually inhaled by the patient only in the inhalation phase, while anyaerosol produced at other times is lost.

Dry powder inhalers, such as metered dose medicament inhalers are wellknown for dispensing medicament to the lungs of a patient. Some previousinhalers have comprised a pressurized aerosol dispensing container,wherein the aerosols contain gas propellants in which the powderedmedicament is suspended. Upon actuation, the aerosol contents areexpelled, through a metering valve, and into the lungs of the patient.

Several types of non-aerosol, breath actuated dry powder inhalers havetherefore been provided. For example, U.S. Pat. No. 5,503,144 to Bacon,shows a breath-actuated dry-powder inhaler. The device includes a drypowder reservoir for containing a dry powdered medicament, a meteringchamber for removal of the powdered medicament from the reservoir indiscrete amounts, and an air inlet for entraining the removed powderedmedicament through a mouthpiece upon patient inhalation.

U.S. Pat. No. 5,458,135 discloses a method and apparatus for producingan aerosolized dose of a medicament for subsequent inhalation by apatient. The method comprises first dispersing a preselected amount ofthe medicament in a predetermined volume of gas, usually air. Thedispersion may be formed from a liquid or a dry powder. The methodrelies on flowing substantially the entire aerosolized dose into achamber that is initially filled with air and open through a mouthpieceto the ambient. After the aerosolized medicament has been transferred tothe chamber, the patient will inhale the entire dose in a single breath.

U.S. Pat. No. 6,065,472 discloses a powder inhalation device comprisinga housing containing a pharmacologically active compound, a conduit withan outlet extending into the housing through which a user can inhale tocreate an airflow through the conduit, a dosing unit for delivering adose of the compound to the conduit and baffles arranged within the saidconduit to aid disintegration of powder agglomerates entrained in saidairflow.

Regardless of whether an aerosol or non-aerosol inhaler is used, it isof utmost importance that particles of the dispensed dry powdermedicament be small enough to ensure the adequate penetration of themedicament into the bronchial region of a patient's lungs duringinhalation. However, because the dry powder medicament is composed ofvery small particles, and often provided in a composition including acarrier such as lactose, non-defined agglomerates or aggregates of themedicament form at random prior to being dispensed. It has thereforebeen found preferably to provide breath-actuated dry powder inhalerswith means for breaking down the agglomerates of medicament ormedicament and carrier before inhalation of the medicament.

The composition and particularly pharmaceutical composition mayoptionally comprise two or more compounds of the present invention. Thecomposition may also be used together with other medicaments within theart for the treatment of related disorders.

The typical dosages of the compounds set forth herein vary within a widerange and depend on many factors, such as the route of administration,the requirement of the individual in need of treatment, the individual'sbody weight, age and general condition.

The compound of formula (I) may be prepared as described in theexperimental section below.

Accordingly, provided is a process of preparing a compound of formula Icomprising the step of reactingbis-(3-deoxy-3-azido-β-D-galactopyranosyl) sulfane with3-fluorophenylacetylene and an amine, such as triethylamine, optionallyin the presence of a catalyst, such as Cu(I), in a solvent, such asN,N-dimethylformamide (DMF), resulting in the compound of formula I. Ina particular embodiment, provided is a process of preparing a compoundof formula I comprising the steps as described in the scheme 1 in theexperimental section. Moreover, the present invention relates to acompound of formula (I) obtainable by the step of reactingbis-(3-deoxy-3-azido-β-D-galactopyranosyl) sulfane with3-fluorophenylacetylene and an amine, such as triethylamine, optionallyin the presence of a catalyst, such as Cu(I), in a solvent, such asN,N-dimethylformamide (DMF), resulting in the compound of formula I,such as obtainable by the steps as described in the scheme 1 in theexperimental section.

The present invention also relates to a method of diagnosing pulmonaryfibrosis in a human subject comprising a) measuring a galectin-3 levelin a body sample from the human subject using a suitable test method, b)comparing the galectin-3 level to a predetermined reference level, andc) determining whether the galectin-3 level is indicative of diagnosingthe subject with pulmonary fibrosis. Such galectin-3 level is typicallythe galectin-3 concentration measured in ng/ml in a body sample such asbody fluid, e.g. blood, plasma, or serum. Such galectin-3 levels mayalso be measured as ng/mg in a body sample consisting of tissue, such aslung tissue.

The term “a predetermined reference level” as used herein means agalectin-3 level which is determined through analysis of a large groupof human subject which are not suffering from pulmonary fibrosis. Suchdetermination of the predetermined reference level have beeninvestigated in several publications, such as US20120220671 andMacKinnon et al., “Regulation of TGF-β1 driven lung fibrosis bygalectin-3”, Am. J. Respir. Crit. Care Med. 185: 537-546, Journal of theAmerican College of Cardiology Vol. xx, No. x, 2012, Ho et al., title:“Galectin-3, a Marker of Cardiac Fibrosis, Predicts Incident HeartFailure in the Community” and Clin Res Cardiol (2010) 99:323-328, Lok etal., title: “Prognostic value of galectin-3, a novel marker of fibrosis,in patients with chronic heart failure: data from the DEAL-HF study”.Based on these studies, the levels have been determined to be within aconcentration range from about 10.0 ng/mL to about 25.0 ng/mLgalectin-3. In some populations said range may be from about 13.0 ng/mLto about 19.2 ng/mL galectin-3.

The indicative level is the level of galectin-3, which to the personskilled in the art, such as a physician, provides such person with atool to set a diagnosis. Typically, the indicative level of galectin-3is at least 22 ng/ml. In a further embodiment the indicative level ofgalectin-3 is at least 25 ng/ml, such as at least 30 ng/ml, at least 40ng/ml, at least 50 ng/ml, at least 60 ng/ml, at least 70 ng/ml.

Furthermore, the present invention relates to a method of predicting aprognosis of pulmonary fibrosis in a human subject comprising a)measuring a galectin-3 level (e.g. concentration) in a body sample fromthe human subject using a suitable test method, and b) determiningwhether the galectin-3 level is indicative of a poor prognosis or notfor the human subject.

The indicative level is the level of galectin-3, which to the personskilled in the art, such as a physician, provides such person with atool to predict the prognosis of the subject. Typically, the indicativelevel of galectin-3 is at least 22 ng/ml. In a further embodiment theindicative level of galectin-3 is at least about 25 ng/ml, such as atleast about 30 ng/ml, at least about 40 ng/ml, at least about 50 ng/ml,at least about 60 ng/ml, at least about 70 ng/ml.

As the tracking of the development of the human subject's disease levelis desired, it is of great importance to develop a method of monitoringthe development, such as improvement or deterioration, or progression ofpulmonary fibrosis, e.g. IPF. It is generally very complicated andcostly to perform clinical trials of novel treatments in these patients.

Accordingly, the present invention relates to a method of monitoringdevelopment or progression of pulmonary fibrosis in a human subject,comprising a) measuring a galectin-3 level in a body sample from thesubject at least two times with sufficient interval(s) to measure aclinically relevant change, b) comparing the galectin-3 level to apredetermined reference level, and repeating steps a) and b) one or moretimes to monitor the development or progression of pulmonary fibrosis inthe human subject.

Whether a change is clinically relevant will be determined by a personskilled in the art, in particular a physician. The time period betweentwo measurements (that is the sufficient interval) is independentlyselected from 2 weeks to 2 years. In individual embodiments such timeperiod between two measurements is selected from 2 weeks, 4 weeks, 1month, 2 months, 3 months 6 months, 1 year, or 2 years.

When the indicative level of galectin-3 is below 22 ng/ml treatment ofpulmonary fibrosis may be stopped, adjusted or put on hold. This istypically determined by the physician.

When the indicative level of galectin-3 is at least about 22 ng/mltreatment of pulmonary fibrosis may be initiated or increased. This istypically determined by the physician. In further embodiments thegalectin-3 level is at least about 25 ng/ml, such as at least about 30ng/ml, at least about 40 ng/ml, at least about 50 ng/ml, at least about60 ng/ml, or at least about 70 ng/ml. In another embodiment the subjecthas a galectin-3 blood concentration determined to be within a targetrange. Typically, such target range is from 10 ng/ml to 70 ng/ml. In aselected or treated human subject, the blood concentration of galectin-3may be determined to be above a minimum threshold, below a maximumthreshold or within a target range defined by a minimum and a maximumthreshold. The minimum threshold may be, for example, more than 10ng/ml; between 10 and 15 ng/ml; between 15 and 20 ng/ml; between 20 and25 ng/ml; between 25 and 30 ng/ml; or be more than 30 ng/ml. The maximumthreshold may be, for example, below 70 ng/ml; below 60 ng/ml; below 40ng/ml; between 30 and 40 ng/ml; between 25 and 30 ng/ml; between 20 and25 ng/ml; or between 15 and 20 ng/ml.

In a further aspect the present invention relates to a method ofmonitoring or predicting exacerbation of symptoms in a human subjectwith pulmonary fibrosis comprising a) measuring a galectin-3 level (e.g.concentration) in a body sample from the human subject using a suitabletest method, b) comparing the galectin-3 level to a predeterminedreference level, b) determine the presence or absence of a galectin-3level indicative of the development or progression of exacerbation ofsymptoms, and if deemed necessary c) repeating steps a) and b) tomonitor or predict the development or progression of the exacerbation ofsymptoms in the human subject.

When the indicative level of galectin-3 is at least about 22 ng/mltreatment of pulmonary fibrosis may be initiated or increased or it maybe decided to monitor the patient more closely to counter the effect ofthe ongoing or eminent exacerbation, if possible. This is typicallydetermined by the physician. In further embodiments the galectin-3 levelis at least about 25 ng/ml, such as at least about 30 ng/ml, at leastabout 40 ng/ml, at least about 50 ng/ml, at least about 60 ng/ml, or atleast about 70 ng/ml. In another embodiment the subject has a galectin-3blood concentration determined to be within a target range. Typically,such target range is from 10 ng/ml to 70 ng/ml. In a selected or treatedhuman subject, the blood concentration of galectin-3 may be determinedto be above a minimum threshold, below a maximum threshold or within atarget range defined by a minimum and a maximum threshold. The minimumthreshold may be, for example, more than 10 ng/ml; between 10 and 15ng/ml; between 15 and 20 ng/ml; between 20 and 25 ng/ml; between 25 and30 ng/ml; or be more than 30 ng/ml. The maximum threshold may be, forexample, below 70 ng/ml; below 60 ng/ml; below 40 ng/ml; between 30 and40 ng/ml; between 25 and 30 ng/ml; between 20 and 25 ng/ml; or between15 and 20 ng/ml.

As it is desired to prevent or reduce exacerbation of symptoms suchprophylactic treatment should be initiated in good time before the levelof galectin-3 reaches 70 ng/ml, thus it is preferred to initiate orincrease prophylactic treatment of exacerbation of symptoms at agalectin level of least 50 ng/ml, such as at least about 60 ng/ml, e.g.at least about 70 ng/ml.

Typically, the pulmonary fibrosis is idiopathic pulmonary fibrosis(IPF).

In a further embodiment the human subject is diagnosed with mild,moderate or aggressive forms of pulmonary fibrosis according to thelevel of galectin-3.

Multimarker analysis can be used to improve the accuracy of diagnosisand monitoring. Expression of markers, such as MMP7 and perDLCO has beenlinked with pulmonary fibrosis (Am J Respir Crit Care Med 185;2012:A6241) When measuring the levels of the above markers, correctionsfor age, gender and concomitant morbidity may be incorporated to improvethe accuracy of diagnosis.

Thus, in a further embodiment in step a) further bio-markers aremeasured which markers are relevant for pulmonary fibrosis, includingmarkers linked to Galectin-3 levels, leading to a more accuratediagnosis, prognosis, and/or monitoring. Typically, such bio-markers areselected from MMP7, perDLCO, KL-6, SP-A, MMP-7, CCL-18, IL13,CC-chemokines, IL10, IL1 receptor antagonist, CCL2, Calgranulin B(S100A9 or MRP14), macrophage migration inhibitory factor (MIF),pro-collagen, or pro-collagen 3 or the presence and frequency of certaincell types in the body sample, such as fibrocytes and T-cellsubpopulations.

The term “a body sample” as used herein means a sample obtained andisolated from a human subject. The body sample may be obtained byvarious known means, such as by biopsy tools, such as a needle biopsytool or a bronchoscope, or by using a syringe.

In a further embodiment the body sample is selected from blood, serum,plasma, broncho-alveolar lavage fluid, and lung tissue.

As explained below several suitable test methods exists and such testmethods are typically selected from an immunoassay, animmunohistochemical assay, a colorimetric assay, a turbidimetric assay,and flow cytometry.

In a further aspect the present invention relates to a method fortreatment of pulmonary fibrosis, such as Idiopathic pulmonary fibrosisin a human subject having a galectin-3 level indicative of pulmonaryfibrosis or exacerbation of symptoms comprising administering to a humansubject a therapeutically effective amount of a galectin-3 inhibitor. Ina particular embodiment the galectin-3 inhibitor is selected from thecompound of formula (I).

In an embodiment the indicative level of galectin-3 is at least about 22ng/ml, such as at least about 25 ng/ml, such as at least about 30 ng/ml,at least about 40 ng/ml, at least about 50 ng/ml, at least about 60ng/ml, at least about 70 ng/ml.

In a further embodiment an additional step of monitoring the subject'sgalectin-3 blood level after the therapy is initiated. In particular,such monitoring is made in accordance with the invention as describedherein.

The present invention provides methods for identification and evaluationof patients with pulmonary fibrosis by measuring the levels of markerssuch as galectin-3, optionally in combination with one or more othermarkers. Many methods for detecting of a protein of interest, with orwithout quantitation, are well known and can be used in the practice ofthe present invention. Such test methods are termed “a suitable testmethod” herein and several useful methods of testing are describedbelow.

Examples of such assays are described below and can include, forexample, immunoassays, chromatographic methods, and mass spectroscopy.Such assays can be performed on any biological sample including, amongothers, blood, plasma, and serum. Accordingly, multiple assays can beused to detect galectin-3, and samples can be analyzed from one or moresources.

Markers can be detected or quantified in a sample with the help of oneor more separation methods. For example, suitable separation methods mayinclude a mass spectrometry method, such as electrospray ionization massspectrometry (ESI-MS), ESI-MS/MS, ESI-MS/(MS)n (n is an integer greaterthan zero), matrix-assisted laser desorption ionization time-of-flightmass spectrometry (MALDI-TOF-MS), surface-enhanced laserdesorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS),desorption/ionization on silicon (DIOS), secondary ion mass spectrometry(SIMS), quadrupole time-of-flight (Q-TOF), atmospheric pressure chemicalionization mass spectrometry (APCI-MS), APCI-MS/MS, APCI-(MS) oratmospheric pressure photoionization mass spectrometry (APPI-MS),APPI-MS/MS, and APPI-(MS) Other mass spectrometry methods may include,inter alia, quadrupole, fourier transform mass spectrometry (FTMS) andion trap. Spectrometric techniques that can also be used includeresonance spectroscopy and optical spectroscopy.

Other suitable separation methods include chemical extractionpartitioning, column chromatography, ion exchange chromatography,hydrophobic (reverse phase) liquid chromatography, isoelectric focusing,one-dimensional polyacrylamide gel electrophoresis (PAGE),two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), or otherchromatographic techniques, such as thin-layer, gas or liquidchromatography, or any combination thereof. In one embodiment, thebiological sample to be assayed may be fractionated prior to applicationof the separation method.

Markers can may be detected or quantified by methods that do not requirephysical separation of the markers themselves. For example, nuclearmagnetic resonance (NMR) spectroscopy may be used to resolve a profileof a marker from a complex mixture of molecules.

A marker in a sample also may be detected or quantified, for example, bycombining the marker with a binding moiety capable of specificallybinding the marker. The binding moiety may include, for example, amember of a ligand-receptor pair, i.e., a pair of molecules capable ofhaving a specific binding interaction. The binding moiety may alsoinclude, for example, a member of a specific binding pair, such asantibody-antigen, enzyme-substrate, nucleic acid-nucleic acid,protein-nucleic acid, protein-protein, or other specific binding pairsknown in the art. Binding proteins may be designed which have enhancedaffinity for a target. Optionally, the binding moiety may be linked witha detectable label, such as an enzymatic, fluorescent, radioactive,phosphorescent or colored particle label. The labeled complex may bedetected, e.g., visually or with the aid of a spectrophotometer or otherdetector, or may be quantified.

Galectin-3 levels can be quantitated by performing an immunoassay. Agalectin-3 immunoassay involves contacting a sample from a subject to betested with an appropriate antibody under conditions such thatimmunospecific binding can occur if galectin-3 is present, and detectingor measuring the amount of any immunospecific binding by the antibody.Any suitable immunoassay can be used, including, without limitation,competitive and non-competitive assay systems using techniques such asWestern blots, radioimmunoassays, immunohistochemistry, ELISA (enzymelinked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, immunodiffusion assays, agglutinationassays, complement-fixation assays, immunoradiometric assays,fluorescent immunoassays and protein A immunoassays.

In a “sandwich” assay, two molecules (“binding moieties”) such asmonoclonal antibodies that specifically bind to non-overlapping sites(epitopes) on galectin-3 are used. Typically, one binding moiety isimmobilized on a solid surface where it binds with and capturesgalectin-3. This first binding moiety is therefore also referred to asthe capture binding moiety. A second binding moiety is detectablylabeled, for example, with a fluorophore, enzyme, or colored particle,such that binding of the second binding moiety to the galectin-3-complexindicates that galectin-3 has been captured. The intensity of the signalis proportional to the concentration of galectin-3 in the sample. Thesecond binding moiety is therefore also referred to as the detectionbinding moiety or label binding moiety. A binding moiety can be any typeof molecule, as long as it specifically binds to a portion of theN-terminus of galectin-3. In a preferred embodiment, the bindingmoieties used are monoclonal anti-galectin-3 antibodies, i.e.,monoclonals raised against or otherwise selected to bind to separateportions of galectin-3.

Such assay procedures can be referred to as two-site immunometric assaymethods, “sandwich” methods or (when antibodies are the binders)“sandwich immunoassays.” As is known in the art, the capture anddetection antibodies can be contacted with the test samplesimultaneously or sequentially. Sequential methods can be accomplishedby incubating the capture antibody with the sample, and adding thelabeled detection antibody at a predetermined time thereafter (sometimesreferred to as the “forward” method). Alternatively, the labeleddetection antibody can be incubated with the sample first and then thesample can be exposed to the capture antibody (sometimes referred to asthe “reverse” method). After any necessary incubation(s), which may beof short duration, to complete the assay, the label is measured. Suchassays may be implemented in many specific formats known to those ofskill in the art, including through use of various high throughputclinical laboratory analyzers or with a point of care or home testingdevice.

In one embodiment, a lateral flow device may be used in the sandwichformat wherein the presence of galectin-3 above a baseline sensitivitylevel in a biological sample will permit formation of a sandwichinteraction upstream of or at the capture zone in the lateral flowassay. See, for example, U.S. Pat. No. 6,485,982. The capture zone maycontain capture binding moieties such as antibody molecules, suitablefor capturing galectin-3, or immobilized avidin or the like for captureof a biotinylated complex. See, for example, U.S. Pat. No. 6,319,676.The device may also incorporate a luminescent label suitable for capturein the capture zone, the concentration of galectin-3 being proportionalto the intensity of the signal at the capture site. Suitable labelsinclude fluorescent labels immobilized on polystyrene microspheres.Colored particles also may be used.

Other assay formats that may be used in the methods of the inventioninclude, but are not limited to, flow-through devices. See, for example,U.S. Pat. No. 4,632,901. In a flow-through assay, one binding moiety(for example, an antibody) is immobilized to a defined area on amembrane surface. This membrane is then overlaid on an absorbent layerthat acts as a reservoir to pump sample volume through the device.Following immobilization, the remaining protein-binding sites on themembrane are blocked to minimize non-specific interactions. Inoperation, a biological sample is added to the membrane and filtersthrough the matrix, allowing any analyte specific to the antibody in thesample to bind to the immobilized antibody. In a second step, a labeledsecondary antibody may be added or released that reacts with capturedmarker to complete the sandwich. Alternatively, the secondary antibodycan be mixed with the sample and added in a single step. If galectin-3is present, a colored spot develops on the surface of the membrane.

The most common enzyme immunoassay is the “Enzyme-Linked ImmunosorbentAssay (ELISA).” ELISA is a technique for detecting and measuring theconcentration of an antigen using a labeled (e.g., enzyme-linked) formof the antibody. There are different forms of ELISA, which are wellknown to those skilled in the art. Standard ELISA techniques aredescribed in “Methods in Immunodiagnosis”, 2nd Edition, Rose andBigazzi, eds. John Wiley & Sons, 1980; Campbell et al., “Methods andImmunology”, W. A. Benjamin, Inc., 1964; and Oellerich, M. (1984), J.Clin. Chem. Clin. Biochem. 22:895-904. A preferred enzyme-linkedimmunosorbent assay kit (ELISA) for detecting galectin-3 is commerciallyavailable (BG Medicine, Waltham, Mass.).

In a “sandwich ELISA,” an antibody (e.g., anti-galectin-3) is linked toa solid phase (i.e., a microtiter plate) and exposed to a biologicalsample containing antigen (e.g., galectin-3). The solid phase is thenwashed to remove unbound antigen. A labeled antibody (e.g., enzymelinked) is then bound to the bound-antigen (if present) forming anantibody-antigen-antibody sandwich. Examples of enzymes that can belinked to the antibody are alkaline phosphatase, horseradish peroxidase,luciferase, urease, and .beta.-galactosidase. The enzyme linked antibodyreacts with a substrate to generate a colored reaction product that canbe measured. Any of the immunoassays described herein suitable for usewith the kits and methods of the present invention can also use anybinding moiety in the place of an antibody.

A detailed review of immunological assay design, theory and protocolscan be found in numerous texts in the art, including Butt, W. R.,Practical Immunology, ed. Marcel Dekker, New York (1984) and Harlow etal. Antibodies, A Laboratory Approach, ed. Cold Spring Harbor Laboratory(1988).

In general, immunoassay design considerations include preparation ofantibodies (e.g., monoclonal or polyclonal antibodies) havingsufficiently high binding specificity for the target to form a complexthat can be distinguished reliably from products of nonspecificinteractions. As used herein, the term “antibody” is understood to meanbinding proteins, for example, antibodies or other proteins comprisingan immunoglobulin variable region-like binding domain, having theappropriate binding affinities and specificities for the target. Thehigher the antibody binding specificity, the lower the targetconcentration that can be detected. As used herein, the terms “specificbinding” or “binding specifically” are understood to mean that thebinding moiety, for example, a binding protein, has a binding affinityfor the target of greater than about 10⁵M⁻¹, more preferably greaterthan about 10⁷M⁻¹.

Antibodies to an isolated target marker which are useful in assays fordetecting heart failure in an individual may be generated using standardimmunological procedures well known and described in the art. See, forexample Practical Immunology, supra. Briefly, an isolated marker is usedto raise antibodies in a xenogeneic host, such as a mouse, goat or othersuitable mammal. The marker is combined with a suitable adjuvant capableof enhancing antibody production in the host, and is injected into thehost, for example, by intraperitoneal administration. Any adjuvantsuitable for stimulating the host's immune response may be used. Acommonly used adjuvant is Freund's complete adjuvant (an emulsioncomprising killed and dried microbial cells and available from, forexample, Calbiochem Corp., San Diego, or Gibco, Grand Island, N.Y.).Where multiple antigen injections are desired, the subsequent injectionsmay comprise the antigen in combination with an incomplete adjuvant(e.g., cell-free emulsion). Polyclonal antibodies may be isolated fromthe antibody-producing host by extracting serum containing antibodies tothe protein of interest. Monoclonal antibodies may be produced byisolating host cells that produce the desired antibody, fusing thesecells with myeloma cells using standard procedures known in theimmunology art, and screening for hybrid cells (hybridomas) that reactspecifically with the target and have the desired binding affinity.

Exemplary epitopes from the N-terminus of galectin-3 include, but arenot limited to, MADNFSLHDALS (SEQ ID NO:1); MADNFSLHDALSGS (SEQ IDNO:2); WGNQPAGAGG (SEQ ID NO:3); YPGAPGAYPGAPAPGV (SEQ ID NO:4);GNPNPQGWPGA (SEQ ID NO:5); YPSSGQPSATGA (SEQ ID NO:6);YPGQAPPGAYPGQAPPGA (SEQ ID NO:7); YPGAPAPGVYPGPPSGPGA (SEQ ID NO:8); andYPSSGQPSATGA (SEQ ID NO:9). Other galectin-3 epitopes, includingnon-linear epitopes, can also be used as targets for detection by ananti-galectin-3 antibody. Exemplary antibodies are discussed in U.S.Patent Publication No. 2010/014954, the entire contents of which areincorporated herein by reference.

Antibody binding domains also may be produced biosynthetically and theamino acid sequence of the binding domain manipulated to enhance bindingaffinity with a preferred epitope on the target. Specific antibodymethodologies are well understood and described in the literature. Amore detailed description of their preparation can be found, forexample, in Practical Immunology, (supra).

In addition, genetically engineered biosynthetic antibody binding sites,also known in the art as BABS or sFv's, may be used to determine if asample contains a marker. Methods for making and using BABS comprising(i) non-covalently associated or disulfide bonded synthetic V.sub.H andV.sub.L dimers, (ii) covalently linked V.sub.H-V.sub.L single chainbinding sites, (iii) individual V.sub.H or V.sub.L domains, or (iv)single chain antibody binding sites are disclosed, for example, in U.S.Pat. Nos. 5,091,513; 5,132,405; 4,704,692; and 4,946,778. Furthermore,BABS having requisite specificity for the marker can be derived by phageantibody cloning from combinatorial gene libraries (see, for example,Clackson et al. Nature 352: 624-628 (1991)). Briefly, phages, eachexpressing on their coat surfaces BABS having immunoglobulin variableregions encoded by variable region gene sequences derived from micepre-immunized with an isolated marker, or a fragment thereof, arescreened for binding activity against the immobilized marker. Phageswhich bind to the immobilized marker are harvested and the gene encodingthe BABS is sequenced. The resulting nucleic acid sequences encoding theBABS of interest then may be expressed in conventional expressionsystems to produce the BABS protein.

Further embodiments of the process are described in the experimentalsection herein, and each individual process as well as each startingmaterial constitutes embodiments that may form part of embodiments.

The above embodiments should be seen as referring to any one of theaspects (such as ‘method for treatment’, ‘pharmaceutical composition’,‘compound for use as a medicament’, or ‘compound for use in a method’)described herein as well as any one of the embodiments described hereinunless it is specified that an embodiment relates to a certain aspect oraspects of the present invention.

All references, including publications, patent applications and patents,cited herein are hereby incorporated by reference to the same extent asif each reference was individually and specifically indicated to beincorporated by reference and was set forth in its entirety herein.

All headings and sub-headings are used herein for convenience only andshould not be construed as limiting the invention in any way.

Any combination of the above-described elements in all possiblevariations thereof is encompassed by the invention unless otherwiseindicated herein or otherwise clearly contradicted by context.

The terms “a” and “an” and “the” and similar referents as used in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless other-wise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. Unless otherwise stated, all exact valuesprovided herein are representative of corresponding approximate values(e.g., all exact exemplary values provided with respect to a particularfactor or measurement can be considered to also provide a correspondingapproximate measurement, modified by “about,” where appropriate).

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise indicated. No language in the specification should beconstrued as indicating any element is essential to the practice of theinvention unless as much is explicitly stated.

The citation and incorporation of patent documents herein is done forconvenience only and does not reflect any view of the validity,patentability and/or enforceability of such patent documents.

The description herein of any aspect or embodiment of the inventionusing terms such as “comprising”, “having”, “including” or “containing”with reference to an element or elements is intended to provide supportfor a similar aspect or embodiment of the invention that “consists of”,“consists essentially of”, or “substantially comprises” that particularelement or elements, unless otherwise stated or clearly contradicted bycontext (e.g., a composition described herein as comprising a particularelement should be understood as also describing a composition consistingof that element, unless otherwise stated or clearly contradicted bycontext).

This invention includes all modifications and equivalents of the subjectmatter recited in the aspects or claims presented herein to the maximumextent permitted by applicable law.

The present invention is further illustrated by the following examplesthat, however, are not to be construed as limiting the scope ofprotection. The features disclosed in the foregoing description and inthe following examples may, both separately and in any combinationthereof, be material for realizing the invention in diverse formsthereof.

EXPERIMENTAL Synthesis ofbis(3-deoxy-3-(3-fluorophenyl-1H-1,2,3-triazol-1-yl)-β-D-galactopyranosyl)sulfane

General Methods.

Melting points were recorded on a Kofler apparatus (Reichert) and areuncorrected. Proton nuclear magnetic resonance (1H) spectra wererecorded on a Bruker DRX 400 (400 MHz) or a Bruker ARX 300 (300 MHz)spectrometer; multiplicities are quoted as singlet (s), doublet (d),doublet of doublets (dd), triplet (t), apparent triplet (at) or apparenttriplet of doublets (atd). Carbon nuclear magnetic resonance (13C)spectra were recorded on a Bruker DRX 400 (100.6 MHz) spectrometer.Spectra were assigned using COSY, HMQC and DEPT experiments. Allchemical shifts are quoted on the d-scale in parts per million (ppm).Low- and high-resolution (FAB-HRMS) fast atom bombardment mass spectrawere recorded using a JEOL SX-120 instrument and low- andhigh-resolution (ES-HRMS) were recorded on a Micromass Q-TOF instrument.Optical rotations were measured on a Perkin-Elmer 341 polarimeter with apath length of 1 dm; concentrations are given in g per 100 mL. Thinlayer chromatography (TLC) was carried out on Merck Kieselgel sheets,pre-coated with 60F254 silica. Plates were developed using 10% sulfuricacid. Flash column chromatography was carried out on silica (Matrex, 60Å, 35-70 μm, Grace Amicon). Acetonitrile was distilled from calciumhydride and stored over 4 Å molecular sieves. DMF was distilled from 4 Åmolecular sieves and stored over 4 Å molecular sieves.

Bis(3-deoxy-3-(3-fluorophenyl-1H-1,2,3-triazol-1-yl)-β-D-galactopyranosyl)sulfane (TD139) was prepared in accordance with the reaction scheme 1below:

Compound (1) (cf. reaction scheme above) is commercial from CarbosynthLimited 8 & 9 Old Station Business Park-Compton-Berkshire-RG20 6NE-UK orsynthesized in three near-quantitative steps from D-galactose, (cf e.g.Li, Z. and Gildersleeve, J. C. J. Am. Chem. Soc. 2006, 128, 11612-11619)

Phenyl2-O-acetyl-4,6-O-benzylidene-1-thio-3-O-trifluoromethanesulfonyl-β-D-galactopyranoside(2)

Compound 1 (10.5 g, 29.2 mmol) was dissolved in dried pyridine (4.73 mL,58.4 mmol) and dried CH₂Cl₂ (132 mL). The reaction mixture was cooled,under stirring, until −20° C. (Ice and NaCl bath 3:1). Slowly and underN₂ atmosphere, Tf₂O (5.68 mL, 33.6 mmol) was added. The reaction mixturewas monitored by TLC (heptane:EtOAc, 1:1 and toluene:acetone, 10:1).When the reaction was complete, AcCl (2.29 mL, 32.1 mmol) was added andkeeping stirring, the temperature was increased to room temperature.This mixture was monitored by TLC too (heptane:EtOAc, 1:1 andtoluene:acetone, 10:1). When it was complete, it was quenched withCH₂Cl₂ and washed with 5% HCl, NaHCO₃ (saturated—hereafter sat) and NaCl(sat). The organic layer was dried over MgSO₄, filtered and concentratedunder reduced pressure.

Phenyl 2-O-acetyl-4,6-O-benzyliden-1-thio-β-D-gulopyranoside (3)

Tetrabutylammonium nitrite (25.3 g, 87.7 mmol) was added to a solutionof compound 2 (15.6 g, 29.2 mmol) in DMF (110 mL) and was kept stirring,under N₂ atmosphere, at 50° C. (The reaction started being purple andturned garnet). The reaction was monitored by TLC (heptane:EtOAc, 1:1and toluene:acetone, 10:1) and quenched with CH₂Cl₂. The mixture waswashed with 5% HCl, NaHCO₃ (sat) and NaCl (sat). The organic layer wasdried over MgSO₄, filtered and concentrated under reduced pressurefollowed by purification by flash chromatography (Eluent heptane:EtOAc,1:1 and heptane:EtOAc, 1:2) and recrystallized from a mixture of EtOAcand Heptane (1:3). ¹H NMR in CDCl₃ δ 7.60-7.57 (m, 2H, Ar), 7.43-7.40(m, 2H, Ar), 7.37-7.34 (m, 3H, Ar), 7.29-7.25 (m, 3H, Ar), 5.50 (s, 1H,PhCH), 5.15 (d, 1H, J=10.29 Hz, H-1), 5.10 (dd, 1H, J=10.27 Hz, 2.85 Hz,H-2), 4.36 (dd, 1H, J=12.49 Hz, 1.4 Hz, H-6), 4.18 (br s, 1H, H-3), 4.08(dd, 1H, J=3.59 Hz, 1.04 Hz, H-6), 4.03 (dd, 1H, J=12.53 Hz, 1.75 Hz,H-4), 3.88 (s, 2H, H-5+OH), 2.12 (s, 3H, OAc).

Phenyl2-O-acetyl-4,6-O-benzylidene-1-thio-3-O-trifluoromethanesulfonyl-β-D-gulopyranoside(4)

Compound 3 (1.00 g, 2.48 mmol) was dissolved in dried CH₂Cl₂ (12.5 mL)and dried pyridine (0.40 mL, 4.96 mmol). The reaction mixture wascooled, under stirring, until −20° C. (Ice and NaCl bath 3:1). Slowlyand under N₂ atmosphere, Tf₂O (0.48 mL, 2.85 mmol) was added. Thereaction mixture was monitored by TLC (heptane:EtOAc, 1:1 andtoluene:acetone, 10:1) and when it was complete, it was quenched withCH₂Cl₂ and washed with 5% HCl, NaHCO₃ (sat) and NaCl (sat). The organiclayer was dried over MgSO₄, filtered and concentrated under reducedpressure until being dry.

Phenyl2-O-acetyl-3-azido-4,6-O-benzylidene-3-deoxy-1-thio-β-D-galactopyranoside(5)

Tetrabutylammonium azide (2.12 g, 7.44 mmol) was added carefully to asolution of compound 4 (1.3256 g, 2.48 mmol) in DMF (10 mL) and was keptstirring, under N₂ atmosphere, at 50° C. The reaction was monitored byTLC (E:H, 1:1) and concentrated under reduced pressure followed bypurification by flash chromatography (Eluent heptane:EtOAc, 2:1 andheptane:EtOAc, 1:1). ¹H NMR in CDCl₃ δ 7.61-7.58 (m, 2H, Ar), 7.44-7.41(m, 2H, Ar), 7.39-7.36 (m, 3H, Ar), 7.30-7.24 (m, 3H, Ar), 5.59 (s, 1H,PhCH), 5.35 (t, 1H, J=9.95 Hz, H-2), 4.73 (d, 1H, J=9.63 Hz, H-1), 4.44(dd, 1H, J=6.24 Hz, 1.60 Hz, H-6), 4.35-4.34 (dd, 1H, J=3.33 Hz, 0.88Hz, H-4), 4.11 (dd, 1H, J=12.48 Hz, 1.67 Hz, H-6), 3.57 (d, 1H, J=1.15Hz, H-5), 3.44 (dd, 1H, J=10.21 Hz, 3.29 Hz, H-3), 2.17 (s, 3H, OAc).

Phenyl 2-O-acetyl-3-azido-3-deoxy-1-thio-β-D-galactopyranoside (6)

Compound 5 (470 mg, 1.1 mmol) was dissolved in 80% acetic acid (75 mL)and the mixture was heated at 60° C. The reaction was monitored by TLC(heptane:EtOAc, 1:1). When the reaction was complete, the mixture wasconcentrated under reduced pressure and heating.

Phenyl 2,4,6-tri-O-acetyl-3-azido-3-deoxy-1-thio-β-D-galactopyranoside(7)

Acetic anhydride (30 mL) was added to a solution of compound 6 (373 mg,1.1 mmol) in dry pyridine (30 mL). The reaction was monitored by TLC(heptane:EtOAc, 1:1) and when it was complete, it was concentrated underreduced pressure. ¹H NMR in CDCl₃ δ 7.54-7.51 (m, 2H, Ar), 7.35-7.30 (m,3H, Ar), 5.46 (dd, 1H, H-4), 5.23 (t, 1H, H-2), 4.73 (d, 1H, H-1), 4.15(d, 2H, H-6, H-6), 3.94 (dt, 1H, H-5), 3.68 (dd, 1H, H-3), 2.18 (s, 3H,OAc), 2.15 (s, 3H, OAc), 2.06 (s, 3H, OAc).

2,4,6-tri-O-acetyl-3-azido-3-deoxy-α-D-galactopyranosyl bromide (8)

Compound 7 (237.4 mg, 560 μmol) was dissolved in dry CH₂Cl₂ (2 mL), andbromine (32 μl, 620 μmol) was added. The reaction was monitored by TLC(heptane:EtOAc, 1:1). When the reaction was complete, a small amount ofcyclopentene was added to the reaction mixture to remove the rests ofBr₂. The mixture was concentrated under reduced pressure and purified byquick Flash chromatography (Eluent: 500 mL heptane:EtOAc, 2:1).

2,4,6-tri-O-acetyl-3-azido-3-deoxy-α-D-galactopyranose-1-isothiouroniumbromide (9)

The sensitive bromide 8 (70.6 mg, 180 μmol) was immediately dissolved indry acetonitrile (1.7 mL) and refluxed with thiourea (13.7 mg, 180 μmol)under N₂ for 4 hours. The reaction was monitored by TLC (heptane:EtOAc,1:1) and when it was complete, the mixture was cooled.

Bis-(2,4,6-tri-O-acetyl-3-azido-3-deoxy-b-D-galactopyranosyl)-sulfane(10)

The sensitive bromide 8 (77.0 mg, 196 μmol) and Et₃N (60 μl, 430 μmol)was added to the last mixture (9). The reaction was monitored by TLC(heptane:EtOAc, 1:1). When it was complete, the reaction mixture wasconcentrated under reduced pressure and without heating. The residue waspurified by flash chromatography (Eluent: heptane:EtOAc, 1:1). ¹H NMR inCDCl₃ δ 5.50 (dd, 2H, H-4,), 5.23 (t, 2H, H-2, H-2′), 4.83 (d, 2H, H-1,H-1′), 4.15 (dd, 4H, H-6, H-6, H-6′, H-6′), 3.89 (dt, 2H, H-5, H-5′),3.70 (dd, 2H, H-3, H-3′), 2.19 (s, 6H, 2OAc), 2.15 (s, 6H, 2OAc), 2.18(s, 6H, 2OAc).

Bis-(3-azido-3-deoxy-β-D-galactopyranosyl)-sulfane (11)

Compound 10 (160 mg, 0.00024 mol) was dissolved in dry MeOH (2.6 mL) anddry CH₂Cl₂ (1.6 mL), and NaOMe (1M, 24 μL, 24 μmol) was added. Thereaction was monitored by TLC (heptane:EtOAc 1:1 and D:M 5:1). When thereaction was complete, the mixture was neutralized with Duolite C436until pH 7, filtered and washed with MeOH. The filtered solution wasconcentrated under reduced pressure. The residue was purified by flashchromatography (Eluent: CH₂Cl₂:MeOH, 5:1) to give pure 11 (74.1 mg,75%). 1H NMR in CDCl₃ δ 4.72 (d, 2H, J=9.7 Hz, H-1, H-1′), 3.95 (br s,2H, H-4, H-4′), 3.84 (t, 2H, J=9.8 Hz, H-2, H-2′), 3.74 (dd, 2H, J=11.47Hz, 7.23 Hz, H-6, H-6′), 3.64 (dd, 2H, J=11.48 Hz, 4.72 Hz, H-6, H-6′),3.60-3.55 (ddd, 2H, 7.15 Hz, 4.67 Hz, 0.93 Hz, H-5, H-5′), 3.36 (dd, 2H,J=10 Hz, 3.05 Hz, H-3, H-3′).

Bis-{3-deoxy-3-[4-(3-fluorophenyl)-1H-1,2,3-triazol-1-yl]-β-D-galactopyranosyl}sulfane(Named TD139)

TD139 was synthesized at ambient temperature by Cu(I)-catalyzedcycloaddition between bis-(3-azido-3-deoxy-β-D-galactopyranosyl)-sulfane(11) and 3-fluorophenylacetylene (3 eq.) with Cu(I) (0.2 eq),triethylamine (2 eq.) in N,N-dimethylformamide (DMF, 100 mL/mmolsulfane). The reaction was monitored with tlc until complete,concentrated and first purified by flash chromatography (Eluent:CH₂Cl₂:MeOH, 8:1), followed by final purification by preparative hplc togive TD139 in 76% yield as a white amorphous solid. ¹H-NMR (CD₃OD, 400MHz) d 8.59 (s, 2H, triazole-H), 7.63 (br d, 2H, 7.6 Hz, Ar—H), 7.57 (brd, 2H, 8.4 Hz, Ar—H), 7.41 (dt, 2H, 6.0 and 8.0 Hz, Ar—H), 7.05 (br dt,2H, 2.4 and 6.4 Hz, Ar—H), 4.93 (dd, 2H, 2.4 and 10.4 Hz, H3), 4.92 (d,2H, 10.4 Hz, H1), 4.84 (2H, 10.4 Hz, H2), 4.18 (d, 2H, 2.4 Hz, H4), 3.92(dd, 2H, 4.2 and 7.6 Hz, H5), 3.84 (dd, 2H, 7.6 and 11.4 Hz, H6), 3.73(dd, 2H, 4.2 and 11.4 Hz, H6); FAB-HRMS m/z calcd for C₂₈H₃₀F₂N₆NaO₈S(M+Na⁺), 671.1712. found, 671.1705.

Model of Bleomycin-Induced Lung Fibrosis

Female C57/Bl6 mice (10-14 weeks old) were anaesthetized with halothane,and bleomycin or saline was administered intratracheally (33 μg in 50 μlof saline) and lungs were harvested on day 26. TD139 was instilled intothe lungs of mice on days 18, 20, 22 and 24 of bleomycin induced lunginjury. Fibrosis was assessed by histological score of collagen stainedlung sections and by total collagen content by Sircol assay.

Mice were treated with bleomycin (bleo) or saline (control) andbleomycin treated mice were treated with 200 mg/kg pirfenidone twicedaily on days 18-24. TD139 was administered intratracheally on days 18,20, 22 and 24. Lungs were harvested on day 26. Results are shown in FIG.1.

Effect on Alveolar Epithelial Cells

Primary alveolar epithelial cells from WT mice were plated and treatedwith TGF-β1 in the presence or absence of 10 μM TD139. FIG. 1D) Cellswere lysed and analyzed for active β-catenin, total β-catenin andβ-actin by western blot.

In conclusion TD139 is a galectin-3 inhibitor and blocked TGF-β-inducedβ-catenin activation in vitro and bleomycin induced lung fibrosis invivo and is believed to represent a novel therapeutic strategy fortreatment of lung fibrosis in mammals, in particular humans.Drug TreatmentMice were divided into the following groups set forth in Table I:Immunohistochemistry

Paraffin-embedded sections of mouse tissue were stained with Masson'strichrome and haemotoxylin and eosin (H&E) as per manufacturer'sinstructions. Sections were processed for immunohistochemistry and thefollowing primary antibodies used: mouse anti-active (ABC) beta-catenin(Millipore) and sections visualized and quantified.

TABLE I Induc- Dosing Adminis- Group tion Treatment Dose days tration 1Control Vehicle N/A 2 Bleo- Vehicle 18, 20, 22 Intratra- mycin and 24cheal 3 Bleo- TD139 10 ug 18, 20, 22 Intratra- mycin and 24 cheal 4Bleo- TD139 3 ug 18, 20, 22 Intratra- mycin and 24 cheal 5 Bleo- TD139 1ug 18, 20, 22 Intratra- mycin and 24 cheal 6 Bleo- TD139 0.1 ug 18, 20,22 Intratra- mycin and 24 cheal 7 Bleo- Pirfenidone 200 mg/kg b.i.d.from oral mycin day 18Determination of Lung Fibrosis and Inflammation

Histological lung inflammation and fibrosis score were carried out inMasson's trichrome stained sections. Inflammation (peribronchiolar,perivascular, and alveolar wall thickness) scored in >5 random fields atmagnification X630 using the following system (peribronchiolar andperivascular, 1=no cells, 2=<20 cells, 3=20-100 cells, 4=>100 cells;alveolar wall thickness, 1=no cells, 2=2-3 cells thick, 3=4-5 cellsthick, 4=>5 cells thick). The combined inflammatory score was the sum ofthese scores. Fibrosis score was evaluated as the area of the sectionpositively stained for collagen (1=none, 2=<10%, 3=<50%, 4=>50%). Onlyfields where the majority of the field was composed of alveoli werescored.

Determination of Lung Collagen by Sircol Assay

Collagen content in the left lung lobe was determined by sircol assay asper manufacturer's instructions. The left lobe was minced in 5 ml of 3mg/ml pepsin in 0.5 M acetic acid and incubated with shaking at 4° C.for 24 h. Cleared lung extract (0.2 ml) was incubated with 0.8 ml sircolreagent for 1 h at room temperature and precipitated collagencentrifuged at 10,000 g for 5 min at 4° C. Pellets solubilised in 1 ml 1M NaOH and absorbance measured at 570 nm alongside collagen standards.

Primary Type II Alveolar Epithelial Cell Isolation

Treated and control mouse type II lung alveolar epithelial cells (AECs)were extracted following a standard method. Briefly, 1 ml of 50 U/mldispase (BD Biosciences) was administered intratracheally into perfusedlungs followed by instillation of 0.5 ml of 1% low melting pointagarose. The agarose within the upper airways was allowed to set on icefor 2 minutes and the lungs were placed in 4 ml 50 U/ml dispase for 45min at room temperature. The lung lobes minus the upper airways werethen dispersed in DMEM containing 50 μg/ml DNAse I (Sigma-Aldrich, UK).The cell suspension was passed through a 100-μm cell strainer and thecells washed in DMEM followed by resuspension in DMEM containing 10%FCS. The cell suspension was plated onto tissue culture plastic for 1 hto allow any contaminated fibroblasts and macrophages to adhere.Non-adherent epithelial cells were counted and cultured for 2 days ontissue culture plastic or cover-slips pre-coated with 5 μg/ml collagen(AMS Biotechnology) and 10 μg/ml fibronectin (Sigma-Aldrich), Cells werewashed three times in PBS before treatment. Epithelial cells were eitherincubated in DMEM containing 10% FCS, 50 U/ml penicillin, 50 μg/mlstreptomycin and 5 μg/ml L-glutamine or transferred to complete mousemedia (DMEM/F-12 containing 0.25% BSA, 10 nM hydrocortisone, 5 μg/mlInsulin-Transferrin-Sodium-Selenite (ITS) and supplemented with 0.1mg/ml sodium succinate, 75 μg/ml succinic acid and 1.8 μg/ml cholinebitartrate).

Western Blotting

Cells were lysed in 25 mM HEPES pH 7.4, 0.3 M NaCl, 1.5 mM MgCl2, 0.2 mMEDTA, 0.5% triton X-100, 0.5 mM dithiothreitol, 1 mM sodiumorthovanadate and protease inhibitors (Boehringer Mannheim, Sussex, UK;prepared as per manufacturers instructions). Lysates equilibrated forprotein using Pierce BCA protein assay reagent (Pierce) and resolved on12% SDS-PAGE gels. Western blot analysis undertaken using the followingprimary antibodies; rabbit anti beta-catenin, (BD Biosciences), rabbitpolyclonal anti-beta-actin antibody (Sigma, UK), mouse anti-active (ABC)beta-catenin (Millipore).

Example 1 Measurement of Galectin-3 Levels in Human Lung Biopsies

Biopsies were sampled from patients with usual interstitial pneumonia(UIP), the most common cause of IPF. Biopsies were fixed in neutralbuffered formalin for 12-24 h prior to embedding in paraffin wax forsectioning. 5 um sections were cut and transferred onto glass slides.Sections were dewaxed in xylene for 10 mins and rehydrated by placingslides for 2 min each in graded ethanol (100%-95%-80%-70%-50%-water)Antigen retrieval was performed by microwaving sections in 0.01M citratepH 6.0 for 15 min. After cooling in running tap water peroxidase wasblocked by incubating in 1% hydrogen peroxide solution for 15 mins.Slides were rinsed in phosphate buffered saline (PBS) and non specificbinding was blocked using serum free protein block and avidin/biotinblocking kit (Vector Laboratories, USA). The sections were incubatedwith mouse monoclonal anti-human galectin-3 clone 9C4 from Novocastra.(diluted to 1:100 in antibody diluent, DAKO, UK) overnight at 4° C.After 3 washes with PBS, sections were incubated with biotinylatedrabbit anti-mouse IgG (H+L) secondary antibody (diluted 1:200 inantibody diluent) for 30 minutes at room temperature. Slides were rinsed3 times with PBS and incubated with 3 drops of avidin:biotinylatedenzyme complex (R.T.U. Vectastain Elite ABC Reagent, PK-7100, VectorLabs, Burlingame, Calif., USA) for 30 minutes followed by liquiddiaminobenzidine (DAB) (Liquid DAB+Substrate Chromogen System, K3468,Dako UK Ltd, Cambridgeshire) in the dark for 10 minutes.

Slides were rinsed 3 times in PBS, counterstained for 30 seconds withMayers haematoxylin (ThermoShandon, UK) and 30 seconds in Scotts tapwater (83 mM MgSO4, 7.1 mM NaHCO3 in tap water), dehydrated throughgraded ethanol (70%, 90%, 100% 2 min each), and cleared in xylene.Slides were mounted using Pertex mounting solution (CellPath HemelHempstead, UK).

Sections were visualized by light microscopy.

Galectin-3 is markedly up-regulated in fibroproliferative areas in thelung of patients with UIP.

Example 2 Method for Measurement of Galectin-3 Levels in Human Serum orHuman Broncho-Alveolar Lavage Fluid

1. Dilute the Capture Antibody to the working concentration in PBSwithout carrier protein. Immediately coat a 96-well microplate6 with 100μL per well of the diluted Capture Antibody. Seal the plate and incubateovernight at room temperature.

2. Aspirate each well and wash with Wash Buffer, repeating the processtwo times for a total of three washes. Wash by filling each well withWash Buffer (400 μL) using a squirt bottle, manifold dispenser, orautowasher. Complete removal of liquid at each step is essential forgood performance. After the last wash, remove any remaining Wash Bufferby aspirating or by inverting the plate and blotting it against cleanpaper towels.3. Block plates by adding 300 μL of Reagent Diluent to each well.Incubate at room temperature for a minimum of 1 hour.4. Repeat the aspiration/wash as in step 2. The plates are now ready forsample addition.Assay Procedure1. Add 100 μL of sample or standards in Reagent Diluent, or anappropriate diluent, per well. Cover with an adhesive strip and incubate2 hours at room temperature.2. Repeat the aspiration/wash as in step 2 of Plate Preparation.3. Add 100 μL of the Detection Antibody, diluted in Reagent Diluent, toeach well. Cover with a new adhesive strip and incubate 2 hours at roomtemperature.4. Repeat the aspiration/wash as in step 2 of Plate Preparation.5. Add 100 μL of the working dilution of Streptavidin-HRP to each well.Cover the plate and incubate for 20 minutes at room temperature. Avoidplacing the plate in direct light.6. Repeat the aspiration/wash as in step 2.7. Add 100 μL of Substrate Solution to each well. Incubate for 20minutes at room temperature. Avoid placing the plate in direct light.8. Add 50 μL of Stop Solution to each well. Gently tap the plate toensure thorough mixing.9. Determine the optical density of each well immediately, using amicroplate reader set to 450 nm. If wavelength correction is available,set to 540 nm or 570 nm. If wavelength correction is not available,subtract readings at 540 nm or 570 nm from the readings at 450 nm. Thissubtraction will correct for optical imperfections in the plate.Readings made directly at 450 nm without correction may be higher andless accurate.

Example 3 Measurement of Galectin-3 Levels in Serum from Patients andControls

Serum was sampled from patients with UIP, patients with non-specificinterstitial pneumonia (NSIP) and aged matched controls. Galectin-3levels were measured using the ELISA method described in example 2.Serum was collected and stored at −80° C. prior to assay. Samples werenormally diluted 1:10 in PBS prior to assay. ELISA was carried out asdescribed in the manufacturers protocol:

Galectin-3 was measured serially (on 2-5 occasions) in the serum of 6patients with stable IPF (UIP). Stable IPF was defined as no significantchange in exercise tolerance, breathlessness or lung function.Galectin-3 was elevated in the serum of patients with IPF (control17.9±0.95 ng/ml n=8, IPF 26.7±4.7 ng/ml n=6, P<0.05) but not in patientswith non-specific interstitial pneumonia (NSIP) (serum concentration14.57±0.84 ng/ml (n=10)).

The serum level of galectin-3 remains remarkably constant over time inthese patients (serum galectin-3 25.5±0.8 ng/ml n=23). We tested 5 serumsamples from patients undergoing an acute exacerbation of IPF. Thesepatients were defined as having an acute exacerbation by decreasedexercise to tolerance, decreased lung function and increasedbreathlessness. In these patients there was a dramatic rise in serumgalectin-3, 73.8±12.2 ng/ml. Furthermore, we identified 2 patients whohad serial galectin-3 measurements prior and during an acuteexacerbation of their IPF. Both patients show stable galectin-3 serumlevels during the period while their lung function was stable. However,during an acute exacerbation when lung function declined there was asharp rise in serum galectin-3.

Example 4 Measurement of Galectin-3 Levels in BAL Fluid from Patientsand Controls

Broncho-alveolar lavage (BAL) fluid was sampled from IPF patients andage matched controls using a standard technique. Briefly, a bronchoscopewas passed through the mouth or nose into the lungs and a small lungsection was flushed with a specified amount of saline. The BAL fluid wascollected and stored at −80° C. The level of Galectin-3 was measuredusing the ELISA method described in Example 2.

Galectin-3 levels were significantly elevated in BAL samples from IPFpatients compared to age matched controls (control 18.8±3.6 ng/ml n=16,IPF 39.7±3.7 ng/ml n=15, P<0.01).

What is claimed is:
 1. A compound of formula (I):

in amorphous form.
 2. The compound of claim 1 wherein said compound is3,3′-Dideoxy-3,3′-bis-[4-(3-fluorophenyl)-1H-1,2,3-triazol-1-yl]-1,1′-sulfanediyl-di-β-D-galactopyranosidein free form.
 3. A composition comprising the compound of claim 1 andoptionally a pharmaceutically acceptable additive.
 4. A method fortreatment of pulmonary fibrosis comprising administering to a mammal inneed thereof an amount of the compound of claim 1 effective to treatsaid pulmonary fibrosis.
 5. The method of claim 4, wherein said compoundis administered by the pulmonary route.
 6. A process of preparing thecompound of claim 1, comprising a step of reactingbis-(3-deoxy-3-azido-β-D-galactopyranosyl) sulfane with3-fluorophenylacetylene and an amine in a solvent, resulting in saidcompound which after purification and isolation provide the amorphousform of the compound of formula (I).
 7. The process of claim 6, whereinthe amine is triethylamine, a catalyst is present and is selected fromCu(I), and the solvent is an organic solvent selected from DMF.